The present invention relates generally to compounds that act as antagonists to the intestinal hormone glucagon-like peptide 1 (GLP-1). More particularly, the invention relates to non-peptide GLP-1 antagonists which have advantageous physical, chemical and biological properties. The GLP-1 antagonists of the present invention inhibit binding of GLP-1 peptide to the GLP-1 receptor and/or prevent the activation of the receptor by bound GLP-1. The invention further relates to a method of inhibiting the binding of GLP-1 to the GLP-1 receptor and a method of inhibiting the activation of the GLP-1 receptor.
GLP-1 is an intestinal hormone released within minutes of food ingestion which potentiates insulin release and aids in the regulation of glucose uptake and metabolism. GLP-1 is derived by post-translational processing of proglucagon and is secreted by the intestinal endocrine L-cells (Fehman et al., 1995, Endocr. Rev. 16:390-410; Thorens et al., 1995, Diabetes Metab. (Paris) 21:311-318). The insulin-trophic effects of GLP-1 make it a useful target in the management of diabetes and other glucose intolerance management problems during critical illness.
Results of recent studies conducted in non-diabetic women aged 59 years suggest that GLP-1 reduces plasma glucose levels primarily by reducing hepatic glucose production and increasing the metabolic clearance rate of glucose through indirectly increasing the insulin-to-glucagon ratio in healthy individuals (Larsson et al., 1997, Acta Physiol. Scand. 160:413-422). Glucose intolerance is a common feature of the aging process; aging has been identified as an etiologic factor for Type II diabetes mellitus.
In a study designed to characterize the abnormalities in beta cells that occur in the aging process, insulin responses were found to be similar in both age groups studied. GLP-1 in conjunction with IVGTT was found to restore the acute insulin response to glucose while increasing the clearance of glucose in the older animals. The conclusion drawn is that an impaired glucose-mediated insulin response is present in the older animals although the animals maintained their insulin responsivity to GLP-1 (Ore et al., 1997, Journal of Gerontology: Biological Sciences 52A(5):B245-B249).
A GLP-1 agonist refers to a compound or agent that mimics the physiological and pharmacological properties of endogenous GLP-1. A GLP-1 antagonist refers to a compound or agent that attenuates the effects of GLP-1 through the ability of these compounds or agents to inhibit GLP-1 peptide binding to the GLP-1 receptor and/or prevent the activation of the receptor by bound GLP-1.
The glucagon-like peptides GLP-1-(7-36)-amide and exendin-4-(1-39) have been identified as GLP-1 agonists. The glucagon-secretin-vasoactive intestinal peptide exendin-(9-39) has been identified as a GLP-1 antagonist (Montrose-Rafizadeh et al., 1997, J. Biol. Chem. 272(34):21201-21206).
Peptide antagonists of peptide hormones are often quite potent. However, the use of peptide antagonists is typically associated with problems due to susceptibility to enzymatic degradation and poor biodistribution, i.e., the inability to be readily transported from the digestive system into the blood stream. Thus, such antagonists have limited effectiveness as drugs since it is difficult to achieve the desired blood levels of peptide antagonists in low dosages. Consequently, there is a need for GLP-1 antagonists, and particularly for non-peptide GLP-1 antagonists.
GLP-1 antagonists have potential to be used therapeutically to increase eating in disorders characterized by cachexia. For example, work by Larsen et al. has shown that the central administration of GLP-1 activates the central CRH-containing neurons of the hypothalamo-pituitary-adrenocortical axis, which may be responsible for feeding behaviors (Larsen et al., 1997, Endocrinology 138(10):4445-4455). Much evidence shows that GLP-1 agonists inhibit food and water intake in rat, and these effects are blocked by the GLP-1 receptor antagonist exendin-(9-39) amide (Navarro et al., 1996, J. Neurochem. 67(5):1982-1991; Tang-Christensen, 1996, Amer. J. Physiol. 271(4 Part 2):R848-856). Exendin-(9-39) alone increases feeding in other rat models (Turton et al., 1996, Nature 379(6560):69-72). In addition, GLP-1 receptor antagonists may be useful in post-prandial hypoglycemia and the dumping syndrome, where there is an exaggerated GLP-1 release (Vecht, 1997, Scand. J. Gastroenterol. Suppl. 223:21-27).
Thus, there is a need for effective non-peptide GLP-1 antagonists useful for the therapeutic regulation of GLP-1 that avoid the in vivo degradation and biodistribution problems exhibited by peptide GLP-1 antagonists.
An object of the present invention is to provide non-peptide GLP-1 antagonists useful as pharmaceuticals. A further object of the invention is to provide methods of synthesizing the compounds and intermediate compounds useful in such syntheses. The compounds of the invention are pharmaceutically superior to peptide compounds since they provide better biodistribution and tolerance to degradation by physiological enzymes.
The invention is directed to GLP-1-antagonizing compounds of the general formula: 
wherein:
R1 is a phenyl or pyridyl group optionally substituted with one or more substituents independently selected from halogen, hydroxyl, nitro, trifluoromethyl, cyano, C1-C6 alkyl, C2-C6 alkenyl, and C1-C6 alkoxy groups;
R2 is: 
xe2x80x83where Rxe2x80x2 is: hydrogen; a hydroxy group; xe2x80x94OR5, where R5 is a C1-C6 alkyl or C2-C6 alkenyl group optionally substituted with a hydroxy group or an amino, C1-C6 alkoxy, cycloalkyl, thioether, heterocycloalkyl, aryl, or heteroaryl group optionally substituted with one or more substituents independently selected from alkyl, hydroxyalkyl, carboxyl, C1-C6 alkoxycarbonyl, oxygen, halogen, and trifluoromethyl groups; or xe2x80x94NR6R7, where R6 and R7 are each independently hydrogen or a C1-C6 alkyl, C2-C6 alkenyl, amino, or imino group optionally substituted with a hydroxy group, a C1-C6 alkoxy group, or an amino, thioether, heterocycloalkyl, aryl, or heteroaryl group optionally substituted with one or more substituents independently selected from oxygen, halogen, trifluoromethyl, and carboxyl groups, or where xe2x80x94NR6R7 forms a 5- or 6-membered heterocyclic ring optionally containing, in addition to the nitrogen heteroatom, a heteroatom selected from O, N, and S;
xe2x80x94(CH2)nxe2x80x94Oxe2x80x94Rxe2x80x3, where n is 1 or 2, and Rxe2x80x3 is hydrogen, a C5-C7 heteroaryl group, or 
xe2x80x83where R8 is hydrogen, a C1-C6 alkyl group, a C3-C6 cycloalkyl group, or a 5- or 6-membered heteroaryl group optionally substituted with one or more substituents independently selected from halogens, methyl, and trifluoromethyl;
xe2x80x94(CH2)pxe2x80x94N(Rxe2x80x3)(Rxe2x80x2xe2x80x3), where p is 1 or 2, Rxe2x80x3 is as defined above, and Rxe2x80x2xe2x80x3 is hydrogen or an alkyl or alkoxy group optionally substituted with a C3-C6 cycloalkyl group optionally substituted with cyano;
xe2x80x94CHxe2x95x90Nxe2x80x94Rxe2x80x3xe2x80x3, where Rxe2x80x3xe2x80x3 is hydrogen, hydroxy, or xe2x80x94OR9, where R9 is an alkyl, cycloalkyl, aryl, or heteroaryl group; or
a 5- or 6-membered heterocyclic ring containing one to three heteroatoms independently selected from O, N, and S, the ring being optionally substituted with one or two substituents independently selected from methyl, methoxymethyl, oxygen, and C1-C6 alkoxy groups;
R3 is hydrogen or a C1-C6 alkyl, C2-C6 alkenyl, or (C1-C3 alkoxy)C1-C3 alkyl group;
or R2 and R3 together with the atoms to which they are bound form a 5- or 6-membered ring containing one or two heteroatoms selected from O, N, and S, the ring being optionally substituted with oxygen, hydroxyl, or a C1-C6 alkyl group optionally substituted with a 5- or 6-membered heterocycloalkyl containing one or two heteroatoms independently selected from O, N, and S; and
R4 is hydrogen or an amino, halogen, hydroxyl, nitro, trifluoromethyl, cyano, C1-C6 alkyl, or C2-C6 alkenyl group.
The invention is also directed to prodrugs, pharmaceutically acceptable salts, pharmaceutically acceptable solvates, and active metabolites of the compounds of the Formula (I).
The GLP-1 antagonists of the present invention inhibit GLP-1 peptide binding to the GLP-1 receptor and/or prevent the activation of the receptor by bound GLP-1. Accordingly, the invention is further directed to a method of inhibiting the binding of GLP-1 to the GLP-1 receptor and a method of inhibiting the activation of the GLP-1 receptor using the inventive compounds.
In accordance with a convention used in the art, 
is used in structural formulas herein to depict the bond that is the point of attachment of the moiety or substituent to the core or backbone structure.
As used herein, the terms xe2x80x9calkyl groupxe2x80x9d is intended to mean a straight- or branched-chain monovalent radical of saturated carbon atoms and hydrogen atoms, such as methyl (Me), ethyl (Et), propyl, isopropyl, butyl, isobutyl, t-butyl, and the like.
The term xe2x80x9calkenyl groupxe2x80x9d refers to a straight- or branched-chain alkene-type radical containing one or more double bonds, such as ethenyl, pentenyl, butenyl, propenyl, and the like.
xe2x80x9cAlkynyl groupxe2x80x9d refers to a straight- or branched-chain alkyne-type radical containing at least one triple bond, such as ethynyl, butynyl, propynyl, pentynyl, hexynyl, and the like.
A xe2x80x9ccycloalkyl groupxe2x80x9d is intended to mean a non-aromatic monovalent monocyclic, bicyclic, or tricyclic radical containing 3 to 14 carbon ring atoms, each of which may be saturated or unsaturated. Illustrative examples of cycloalkyl groups include the following moieties: 
A xe2x80x9cheterocycloalkyl groupxe2x80x9d is intended to mean a non-aromatic monovalent monocyclic, bicyclic, or tricyclic radical, which is saturated or unsaturated, containing 3 to 18 ring atoms, which includes 1 to 5 heteroatoms selected from nitrogen, oxygen, and sulfur. Illustrative examples of heterocycloalkyl groups include the following moieties, where R is any suitable substituent: 
An xe2x80x9caryl groupxe2x80x9d is intended to mean an aromatic monovalent monocyclic, bicyclic, or tricyclic radical containing 6 to 18 carbon ring atoms. Illustrative examples of aryl groups include the following moieties: 
A xe2x80x9cheteroaryl groupxe2x80x9d is intended to mean an aromatic monovalent monocyclic, bicyclic, or tricyclic radical containing 4 to 18 ring atoms, including 1 to 5 heteroatoms selected from nitrogen, oxygen, and sulfur. Illustrative examples of heteroaryl groups include the following moieties: 
A xe2x80x9cheterocyclexe2x80x9d is intended to mean a heteroaryl or heterocycloalkyl group.
An xe2x80x9cacyl groupxe2x80x9d is intended to mean a xe2x80x94C(O)xe2x80x94R radical, where R is a carbonxe2x80x94, oxygenxe2x80x94, nitrogenxe2x80x94, or sulfurxe2x80x94linked substituent.
A xe2x80x9csulfonyl groupxe2x80x9d is intended to mean an xe2x80x94SO2R radical, where R is a carbon-, oxygen-, or nitrogen-linked substituent.
An xe2x80x9camino groupxe2x80x9d is intended to mean an xe2x80x94NH2 radical or a primary, secondary, or tertiary amine radical (e.g., NHRa, where Ra is an alkyl group; and xe2x80x94NRaRb, where Ra and Rb are each independently an alkyl group).
An xe2x80x9ciminoxe2x80x9d substituent refers to a substituent containing a carbonxe2x80x94nitrogen double bond, for example, 
An xe2x80x9calkoxy groupxe2x80x9d is intended to mean the radical xe2x80x94ORa, where Ra is an alkyl group. Exemplary alkoxy groups include methoxy, ethoxy, propoxy, and the like.
An xe2x80x9calkoxycarbonyl groupxe2x80x9d is intended to mean the radical xe2x80x94C(O)ORa, where Ra is an alkyl group.
The term xe2x80x9cthioetherxe2x80x9d refers to alkylthio, arylthio, and heteroarylthio groups. An xe2x80x9calkylthio groupxe2x80x9d is intended to mean the radical xe2x80x94SRa, where Ra is an alkyl group. An xe2x80x9carylthio groupxe2x80x9d is intended to mean the radical xe2x80x94SRc, where Rc is an aryl group. A xe2x80x9cheteroarylthio groupxe2x80x9d is intended to mean the radical xe2x80x94SRd, where Rd is a heteroary. group.
An xe2x80x9caryloxy groupxe2x80x9d is intended to mean the radical xe2x80x94ORc, where Rc is an aryl group. A xe2x80x9cheteroaryloxy groupxe2x80x9d is intended to mean the radical xe2x80x94ORd, where Rd is a heteroaryl group.
The term xe2x80x9csubstituentxe2x80x9d or xe2x80x9csuitable substituentxe2x80x9d is intended to mean any chemically suitable substituent that may be recognized or selected, such as through routine testing, by those skilled in the art. Illustrative examples of suitable substituents include hydroxy (xe2x80x94OH), halogens, oxo groups, alkyl groups, acyl groups, sulfonyl groups, mercapto groups, alkylthio groups, alkoxy groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, carboxy (xe2x80x94C(O)OH), amino groups, carbamoyl (xe2x80x94C(O)NH2), aryloxy groups, heteroaryloxy groups, arylthio groups, heteroarylthio groups, and the like.
The term xe2x80x9coptionally substitutedxe2x80x9d is intended to indicate that the specified group is unsubstituted or substituted by one or more suitable substituents, unless the optional substituents are expressly specified, in which case the term indicates that the group is unsubstituted or substituted with the specified substituents. As defined above, various groups may be unsubstituted or substituted (i.e., they are optionally substituted) unless indicated otherwise herein (e.g., by indicating that the specified group is unsubstituted).
A xe2x80x9cprodrugxe2x80x9d is intended to mean a compound that is converted under physiological conditions or by solvolysis or metabolically to a specified compound that is pharmaceutically active.
A xe2x80x9cpharmaceutically active metabolitexe2x80x9d is intended to mean a pharmacologically active product produced through metabolism in the body of a specified compound.
A xe2x80x9csolvatexe2x80x9d is intended to mean a pharmaceutically acceptable solvate form of a specified compound that retains the biological effectiveness of such compound. Examples of solvates include compounds of the invention in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.
A xe2x80x9cpharmaceutically acceptable saltxe2x80x9d is intended to mean a salt that retains the biological effectiveness of the free acids and bases of the specified compound and that is not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, xcex3-hydroxybutyrates, glycollates, tartrates, methanesulfonates (mesylates), propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.
The action of GLP-1 is antagonized by the 9H-xcex2-carboline compounds of the general Formula (I): 
wherein R1, R2, R3, and R4 are as defined above. The invention is also directed to prodrugs, pharmaceutically acceptable salts, pharmaceutically acceptable solvates, and active metabolites of such compounds.
In a preferred embodiment, R1 is a phenyl group substituted with one or more groups selected from halogen, hydroxyl, nitro, trifluoromethyl, and cyano.
Also preferred are compounds where R2 is 
where Rxe2x80x2 is as defined above and incorporates a hydrogen-bond acceptor substituent that can, through normal conformational variations, assume a position 3-5 xc3x85 from the carbonyl group. As used herein, xe2x80x9chydrogen-bond acceptor substituentxe2x80x9d refers to a substituent that includes an N or O capable of forming a hydrogen bond with a hydrogen-bond donor such as xe2x80x94OH or xe2x95x90NH. Exemplary hydrogen-bond acceptor substituents include moieties containing a group such as 
Exemplary R2 groups of this type include 
As known in the art, the shorthand designation 
is used herein to depict xe2x80x94CH3.
Also preferred are compounds wherein R3 is hydrogen or methoxymethyl.
In a further preferred embodiment, R1 is 2,5-dichlorophenyl or 3,5-dinitrophenyl.
In another preferred embodiment, R2 and R3 together with the atoms to which they are bound form a 5- or 6-membered lactone or lactam ring.
In yet another preferred embodiment, R2 is selected from: 
In another preferred embodiment, the 5- or 6-membered ring formed by R2 and R3 and the atoms to which they are bound is selected from: 
Especially preferred compounds represented by the above general Formula (I) include the following: 
In addition, the present invention is directed to precursors, building blocks, and intermediates that are useful in preparing the compounds of Formula (I). The examples illustrate specific precursors, building blocks, and intermediates within the scope of the present invention. In particular, the following compounds can be used to synthesize certain compounds within the scope of the present invention: 
The compounds of the present invention include prodrugs, pharmaceutically acceptable salts, pharmaceutically acceptable solvates, and active metabolites of compounds of the Formula (I). The salts of the compounds are pharmaceutically acceptable salts derived from inorganic or organic acids as defined above.
The invention further comprises active metabolites and prodrugs of the compounds of Formula (I). Active metabolites of the present invention have undergone modification to their chemical structure resulting from being acted on by biotransformation reactions of drug metabolizing enzymes in various organs of the body. Prodrugs are compounds that, through these various biotransformation reactions, are metabolically converted in vivo from a precursor compound to a compound of Formula (I). Examples of prodrugs include biohydrolyzable esters and amides.
Some compounds of the invention described herein contain one or more centers of asymmetry and may thus give rise to enantiomers, diastereoisomers, and other stereoisomeric forms. The present invention is meant to include all such possible stereoisomers as well as their racemic and optically pure forms. Optically active (R) and (S) isomers may be prepared using chiral synthons, chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds, both E and Z geometric isomers are comprehended.
The chemical formulae referred herein may exhibit the phenomenon of tautomerism. As the formulae drawings within this specification can only represent one of the possible tautomeric forms, it should be understood that the invention encompasses any tautomeric form which can be generated by employing the tools disclosed and is not limited to any one tautomeric form utilized within the formulae drawings.
Pharmaceutical Compositions and Methods of Treatment
The pharmaceutical compositions of this invention comprise an effective amount of a compound of Formula (I) and an inert pharmaceutically acceptable carrier or diluent. An xe2x80x9ceffective amountxe2x80x9d of a compound of Formula (I) is determined to be a GLP-1 antagonistic amount, which is a concentration of the compound where the binding and/or activation of the GLP-1 receptor is inhibited. Such an amount provides therapeutic benefits for the regulation of the insulin trophic effects associated with GLP-1 binding.
The inventive pharmaceutical compositions are prepared in dosage unit form appropriate for administration to a patient in need of treatment of a disease or condition mediated by GLP-1 inhibition. Appropriate forms of administration include (but are not limited to) oral, parenteral, intravenous, intramuscular, and transdermal methods that are generally known in the art.
The compositions may be prepared by combining an effective amount of the compound of the Formula (I) with known pharmaceutical carriers or diluents according to conventional procedures. These procedures may involve mixing, granulating, compressing or dissolving the ingredients as appropriate for the desired preparation.
The pharmaceutical carrier employed may be, for example, either a solid or liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may include time-delay material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate or the like.
A variety of pharmaceutical forms can be employed. Thus, if a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge. The amount of solid carrier may vary, and preferably will be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation may be in the form of syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampoule or vial or nonaqueous liquid suspension.
To obtain a stable water-soluble dose form, a pharmaceutically acceptable salt of a compound of Formula (I) is dissolved in an aqueous solution of an organic or inorganic acid, such as 0.3 M solution of succinic acid, or preferably, citric acid. If a soluble salt form is not available, the compound of Formula (I) is dissolved in one or more suitable cosolvents. Examples of suitable cosolvents include (but are not limited to) alcohol, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin and the like in concentrations ranging from 0-60% of the total volume.
The composition may also be in the form of a solution of a salt form of the active ingredient in an appropriate aqueous vehicle, such as water or isotonic saline or dextrose solution.
It will be appreciated that the actual dosages of the Formula (I) compounds used in the compositions of this invention will be selected according to the particular complex being used, the particular composition formulated, the mode of administration and the particular site, and the host and condition being treated. Optimal dosages for a given set of conditions can be ascertained by those skilled in the art using conventional dosage-determination tests. For oral administration, e.g., the dose generally employed is from about 0.001 to about 1000 mg/kg body weight, with courses of treatment repeated at appropriate intervals.
Synthesis Methods
The following synthesis protocols refer to preferred intermediate compounds and final products identified in the synthesis schemes or elsewhere in the specification. The preparation of compounds of the present invention is described in detail using the following general and specific examples. Occasionally, the reaction may not be applicable as described to each compound included within the disclosed scope of the invention; the compounds for which this occurs will be readily recognized by those skilled in the art. In all such cases, the reactions can be successfully performed by routine modifications within the level of ordinary skill in the art (e.g., by reference to teachings in the art, including those cited herein), such as by appropriate protection of interfering groups, by changing to other conventional reagents, or by routine changes to reaction conditions. Alternatively, other reactions disclosed herein or otherwise conventional will be applicable to the preparation of the corresponding compounds of the invention. In the preparative methods described below: all starting materials are known, available or readily prepared from known starting materials; all temperatures are set forth in degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.
Reagents were purchased from commercial suppliers such as Aldrich Chemical Company or Lancaster Synthesis Ltd. and were used without further purification unless otherwise indicated. Tetrahydrofuran (THF) and N, N-dimethylformamide (DMF) were purchased from Aldrich in sealed bottles and used as received. All solvents were purified using standard methods known to those skilled in the art, unless otherwise indicated.
Biological Assays
In general, the activity of the non-peptide antagonists of the present invention may be determined using a variety of assays and techniques. The GLP-1 antagonists of the present invention inhibit the binding of GLP-1 to its receptor and/or inhibit receptor activation by bound GLP-1. Thus, binding affinity studies are useful to assess the antagonistic activity of the compounds of the present invention. Binding affinity may be determined, for example, by displacement of a ligand bound to the receptor, where the ligand is labeled with a detectable label. In particular, one having ordinary skill in the art could conduct an in vitro binding study to calculate the specific binding affinity of the compounds of the present invention to the GLP-1 receptor by pretreating cells with the compounds and then challenging the pretreated cells with radioactively labeled GLP-1.
Additionally, one having ordinary skill in the art would appreciate that the activation of the GLP-1 receptor can be measured by determining the intracellular cAMP levels measured in cells treated with the compounds of the present invention. See, e.g., Montrose-Rafizadeh et al., 1997, J. Biol. Chem. 272(34):21201-21206. After treatment with the compounds of the present invention, the cells are challenged with GLP-1 and the intracellular cAMP levels are determined. Antagonistic activity would be represented by decreased levels of cAMP relative to a non-treated control.
In addition to these biological assays, other peripheral assays are suitable to determine the antagonistic activity of the compounds of Formula (I). For example, known assays for determining GLP-1 activity include ingestion bioassays and ANG II-stimulated thirst assays (Tang-Christensen et al., 1996, Amer. J. Physiol. 271(4 Pt 2):R848-R856), and lipolysis assays (Montrose-Rafizadeh et al., 1997, J. Cell Phys. 172(3):275-283).
Based upon the foregoing assays, one having ordinary skill in the art could determine the effectiveness of the compounds of the present invention to inhibit the binding and/or activation of the GLP-1 receptor by GLP-1. Furthermore, such studies would be useful in assessing the effective amounts of the compounds of the present invention to inhibit GLP-1 activity.
General Examples
Method A: General Procedures for N-alkylation 
In the compounds set forth above, R1, R4, and R5 are as defined above.
To a solution of alkyl halides in DMF (1 equiv) is added a DMF solution of substituted methyl 9H-xcex2-carboline-3-carboxylate (1 equiv) and a suspension of sodium hydride in DMF (xcx9c1 equivalent of 60% NaH in oil) is added to the mixture. The mixture is covered, briefly agitated and shaken briefly every 15 minutes for approximately one hour. The DMF is removed in vacuo.
Method B: N-Alkylation 
In the above compounds, Z represents xe2x80x94CH2R1, which is as defined above.
A mixture of a methyl 9H-xcex2-carboline-3-carboxylate or derivative (5.0 mmol) and sodium hydride (0.20 g of 60% in oil, 5.0 mmol) is treated with dry DMF (11 mL) under nitrogen. After stirring for 15 minutes, gas evolution is essentially complete, affording a light brown, nearly clear solution of the sodium salt of the xcex2-carboline (xcx9c0.40 M), with a trace of solid material still remaining. This solution is similarly prepared by addition of the solid xcex2-carboline to a slurry of sodium hydride in DMF, or by addition of sodium hydride to a slurry/solution of the xcex2-carboline in DMF. Cooling to 0xc2x0 C. is necessary when the reaction is carried out on a larger scale (40-80 mmol).
To the solution of the sodium salt is added a solution of an alkyl halide in DMF (5 mL of 1.0 M, 5.0 mmol, 1 equiv), resulting in a slight exotherm. After stirring at room temperature for 2-24 hours, the DMF was removed in vacuo, and the residue partitioned between water and ethyl acetate. The ethyl acetate phase is separated, dried over Na2SO4, filtered, concentrated in vacuo, and the residue crystallized from ethyl ether, ethyl acetate/ethyl ether, or ethyl acetate/petroleum ether.
Alternatively, the products are purified by chromatography on silica gel using 95:5 diethyl ether/8M NH3xe2x80x94CH3OH or a gradient elution of 90:10 trichloromethane (CHCl3)/2M NH3-CH3OH in CHCl3, or reverse-phase preparative HPLC, followed by recrystallization from ethyl ether or ethyl acetate/petroleum ether.
Method C: Esterification from carboxylic acid imidazolide 
In the compounds set forth above, R5 is as defined above.
Esterification of acids via 3-(1-imidazolylcarbonyl)-9-(2,5-dichlorobenzyl)-9H-xcex2-carboline may be conducted by the following method (Staab, H. A., ACIEE 1962, 1:351). To a solution of an alcohol (125 xcexcL of 0.40 M, 0.050 mmol) in DME (1,2-dimethoxyethane) is added a solution of a 3-(1-imidazolylcarbonyl)-9-(2,5-dichlorobenzyl)-9H-xcex2-carboline (125 xcexcL of 0.40 M, 0.050 mmol, 1 equiv) in DMF, followed by a solution of imidazolyl sodium (25 xcexcL of 0.1 M, 0.0025 mmol, 5 mol %) in DMF. The latter is freshly prepared from imidazole and sodium hydride. The resulting mixture is briefly agitated and heated at 50xc2x0 C. for 18-24 hours. The solvents are removed in vacuo, and the product is isolated from the residue by preparative HPLC.
Method D: Esterification of an Alcohol 
In the compounds set forth above, R8 is as defined above.
Esterification of an alcohol such as [9-(2,5-dichlorobenzyl)-9H-xcex2-carbolin-3-yl]methanol (see above) may be conducted by the following method. A solution of [9-(2,5-dichlorobenzyl)-9H-xcex2-carbolin-3-yl]methanol (100 xcexcL of 0.5 M, 0.05 mmol) in DME is treated with a solution of an acid chloride (100 xcexcL of 0.5 M, 0.05 mmol) in DCE (1,2-dichloroethane), and the mixture agitated briefly. A solution of triethylamine (100 xcexcL of 1.0 M, 0.1 mmol, 2equiv) in DME is added, and the mixture is agitated again and allowed to stand at room temperature overnight. The volatiles are removed in vacuo, and the product is isolated from residue by preparative HPLC.
Method E: Esterification of a Carboxylic Acid 
A carboxylic acid such as 9H-xcex2-carboline-3-carboxylic acid (see above) may be esterified according to the present invention as follows. 9H-xcex2-Carboline-3-carboxylic acid is stirred with excess SOCl2 at room temperature overnight. The excess SOCl2 is removed in vacuo and the resulting crude acid chloride is dissolved in CHCl3. To this solution is added Et3N (3 equiv) and an alcohol (5 equiv), and the resulting mixture is stirred at room temperature overnight. The reaction mixture is subjected to an aqueous workup, and the residue from the organic phase is chromatographed on silica gel (CH2Cl2) affording the ester.